Chromatin structure is critical to the regulation of eukaryotic gene expression. The basic unit of chromatin, the nucleosome, regulates transcription by limiting the accessibility of transcriptional factors to specific DNA sequences within promoters. A better biochemical understanding of how eukaryotic genes are regulated will require reconstituted transcription in the context of genomic DNA packaged with properly organized nucleosomes. Our lab recently used the latest in DNA sequencing technology to show that the in vivo pattern of nucleosome positioning can be recapitulated in vitro across the S. cerevisiae genome by chromatin remodelers. Here we propose to develop a genome- wide immobilized template assay that will allow us to ask fundamental questions regarding nucleosome dynamics, transcription factor occupancy, and RNA transcription on a genome-wide scale in the context of proper chromatin structure. With this assay aspects of gene expression regulation will be investigated in regards to transcription factor binding and the transcriptional requirements of histone modifications. These studies are intended to shed light on two fundamental questions in gene regulation: Can transcription factors intrinsically access their binding sites in native chromatin, or is the cooperation of remodelers needed? Does the physiological pattern of active histone modifications arise before or after transcription occurs? The development of this assay will lead to a technique in which this in vitro system will be programmed to contain specific aberrant chromatin states as are seen in disease states in order to determine how these changes in chromatin structure lead to the development of cancer, along with cardiovascular, neurodegenerative, and autoimmune diseases.